Lateral Flow Test Kit and Method for Detecting an Analyte

ABSTRACT

A method and device for detecting analytes in a test sample. Embodiments include methods for quantitatively detecting analytes within a range of concentrations. In an embodiment the method includes a lateral flow test strip with multiple test areas for capturing a labeled receptor to provide a detectable signal.

REFERENCE TO PRIOR APPLICATIONS

This application is based on and claims priority from U.S. Provisionalpatent Application Ser. No. 60/654,292, filed Feb. 18, 2005, which ishereby incorporated by reference. This application is a divisionalapplication of U.S. patent application Ser. No. 11/883,784, filed Aug.6, 2007.

FIELD OF APPLICATION

The application relates to detecting an analyte, such as a smallmolecule, in a sample using a solid support such as a lateral flow typetest strip.

BACKGROUND

Tests to detect analytes in samples are known in the art. Some examplesare described in U.S. Pat. No. 5,985,675, issued Nov. 16, 1999; U.S.Pat. No. 6,319,466, issued Nov. 20, 2001; U.S. patent application Ser.No. 10/289,089, filed Nov. 6, 2002 (based on U.S. ProvisionalApplication 60/332,877, filed Nov. 6, 2001); U.S. patent applicationSer. No. 09/961,998, filed Sep. 24, 2001, and U.S. patent applicationSer. No. 10/993,340, filed Nov. 19, 2004, all of which are incorporatedherein by reference in their entirety.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a side view of an embodiment having two test areas 9, 4 and acontrol area/zone 5.

FIG. 2A are graphs relating intensity of color development on a teststrip (Y axis), with 2 test areas (bottom and middle) and a control area(top), to aflatoxin concentration in samples (X axis).

FIG. 2B are graphs of the results from the same experiments as in FIG.2A with additional concentrations included.

FIG. 3A are graphs relating intensity of color development on a teststrip (Y axis), with 1 test area (bottom) and a control area (top), toaflatoxin concentration in samples (X axis).

FIG. 3B are graphs similar to 3A and showing that reducing theconcentration of capture agent in the top area reduces the intensity ofcolor development at the top line but does not significantly change thespread between results at various concentrations.

FIG. 4 is a graph comparing results from a single test area/control areatest for aflatoxin (2 line ave) with a two test area/single control areatest for aflatoxin (3 line ave), where only the first test area, of thetwo test area test, was used to calculate results. This graphdemonstrates that even when the second test area is not used tocalculate results, the added binding capacity created by the presence ofthe second test area can help increase the detection range of the test.

SUMMARY

One aspect involves a method for the analysis of a liquid sample for thepresence of an analyte in which the sample is contacted with a receptorto form a mobile phase. The receptor can be characterized by an abilityto bind to analyte to provide, in the mobile phase, a receptor-analytecomplex.

The mobile phase contacts, or is put into contact with, a first testarea on a solid support. The solid support can be configured to allowthe mobile phase to flow from the first test area to a second test areaon the solid support and, if a control zone is included, to the controlzone. The first test area can include a capture agent immobilized on thesolid support. The first test area capture agent will have greaterbinding affinity to the receptor than to the receptor-analyte complex.As a result of that differential in binding affinity, captured receptorin the test area will decrease as sample analyte concentrationincreases. The second test area can also include a capture agentimmobilized on the solid support. As with the first area capture agent,the second test area capture agent will have greater binding affinity tothe receptor than to the receptor-analyte complex. The capture agent canbe the same in each of the test areas and at the same or differentconcentrations in each area. The capture agents can also be different,for example with different binding characteristics to the receptor.

The receptor can be labeled with a label, such as a colored particle,that can be detected when the receptor is bound to the solid support viacapture by the capture agent immobilized on the solid support. Theintensity of the detectable signal, for example a visible signal, at thefirst and second test areas can be measured to determine a result. In aninhibition style test the strength (intensity) of the signals areinversely related to the concentration of analyte in the sample. Thesignal intensities can be observed visually or measured by an electronictest instrument. For example the intensity at each of the two test areascan be summed to determine a result that can relate to the concentrationof an analyte in the sample.

In another embodiment a control zone is included. The control zone caninclude a control zone capture agent on the solid support. The controlzone capture agent can have affinity to the receptor that is equivalentto the control zone capture agent's affinity to the receptor-analytecomplex. When the control zone capture agent has affinity to thereceptor that is equivalent to its affinity to the receptor-analytecomplex, the signal in the control zone will increase as analyteconcentration in the sample increases. The control zone can be comparedto the test areas to determine the result. In an example a digitalreading, related to signal intensity, is provided by an electronicinstrument that calculates the intensity differences between the firstand second test areas and the control zone and sums the results toarrive at the approximate concentration of analyte in the sample, forexample by comparing the test result to a predetermined value. A varietyof reading methods can be employed depending on the label used. When thelabel is a colored particle, such as a gold particle, the reader can bea reflectance reader.

The solid support can be a lateral flow test strip with a stationaryphase membrane on which the test and control zones are placed. Themembrane can allow lateral capillary flow of the sample from the firstend to the second end and has the test areas thereon.

In some embodiments the receptor is labeled prior to application to thetest strip. In other embodiments the label can be contracted with thereceptor during test operation. The receptor can be an antibody such asan antibody to an analyte such as a mycotoxin, for example, aflatoxin orother small molecules such as antibiotics.

Control zone capture agents can include antibody binding proteins suchas protein A and/or antibodies such as anti-species antibody. Thecapture agent can be immobilized directly on the solid support orthrough attachment to a carrier protein. The control zone can be usedfor comparison to the test areas and also can be used to inform the userthat the mobile phase has flowed through the test strip and/or that thetest is functioning properly. In that sense the control zone can be usedas a flow control. The control zone can also be involved in a reactionthat is independent of the analyte-receptor reaction. In that way thecontrol zone can provide, if desired, a consistent signal that does notvary with concentration of analyte. Such an independent reaction caninclude providing a separate labeled receptor with affinity only to thecontrol zone capture agent.

DESCRIPTION

Lateral flow type test strips are an example of a test utilizing a solidsupport to capture a detectable signal. Lateral flow tests can be usedto detect one or more substances (analytes) in a fluid sample. Lateralflow strips generally include a stationary phase and a mobile phase. Thestationary phase can include various reagents immobilized on the teststrip. The mobile phase can include the fluid sample that flows overand/or through the test strip. The mobile phase can also include avariety of reagents. As the mobile phase flows it also can carry with itreagents that may be reconstituted from the strip. The mobile phase canalso include solutions, such as dilution buffer. As an alternative toreconstitution from the strip, mobile phase reagents can be mixed withthe sample prior to application of the sample to the strip.

Reaction of a substance in the mobile phase with a stationary phasereagent can generate a detectable signal. The stationary phase reagents,sometimes referred to as capture agents, can be immobilized on the stripso that they capture substances from the mobile phase. The signal can begenerated by a reagent from the mobile phase, often referred to as alabel, which attaches to one or more of the stationary phase reagents.Various suitable labels include chromogens, catalysts, fluorescentcompounds, chemiluminescent compounds, radioactive labels, magneticbeads or magnetic particles, enzymes or substrates, vesicles containingsignal producing substances, colorimetric labels, direct visual labelsincluding colloidal metallic and metallic and non-metallic coloredparticles, dye particles, or organic polymer latex colored particles.

To detect the presence or absence of an analyte test strips can bedesigned to provide a signal that can be observed visually, such ascolor changes or color differences on the test strip. The signal canalso be observed, measured and/or interpreted visually or with a reader.A variety of readers are appropriate including spectrophotometers, LCDcameras, reflectance readers, luminometers, fluorometers, scintillationcounter, magnetic detectors and other instruments capable of reading,measuring and/or interpreting changes on a lateral flow test strip. Onesuch instrument is described in U.S. Pat. No. 6,124,585, issued Sep. 26,2000, hereby incorporated by reference. Another such instrument is aROSA Reader (ROSA is a registered trademark of Charm Sciences, Inc.,Lawrence, Mass.).

Presence or absence tests, known in the art as qualitative tests,provide a yes or no result. Tests that detect the presence or absence ofa target analyte above or below a certain threshold level are known assemi-quantitative tests. Tests that determine that a target analyte ispresent at a particular concentration, or within a range ofconcentrations, are known as quantitative tests. Although quantitativetests may determine that an analyte is present within a range ofconcentrations or at a particular level the results also have a range oferror. For example, a result that an analyte is present at 25 parts perbillion (ppb) can be within a range of acceptable error, for example aresult of 25 ppb can mean that analyte is present in a range of 20-30ppb. Such a test is referred to as quantitative. Tests that providequantitative results within a limited range are also described asquantitative. For example, results may indicate that an analyte ispresent within a range of 0 to 100 ppb but above 100 ppb no conclusioncan be drawn other than that the result is above 100 ppb.

Quantitative results are sometimes preferred or required. For example,the United States Department of Agriculture, Grain Inspection, Packersand Stockyard Administration, Federal Grain Inspection Service, ProgramNotice FGIS-PN-04-15, dated Jun. 16, 2004, describes performancecriteria for test kits. Criteria for aflatoxin in corn includequantitative detection at 5 ppb, 10 ppb, 20 ppb and 100 ppb totalaflatoxin (B1+B2+G1+G2). One of the herein described embodimentsincludes a lateral flow test strip that meets those criteria. Variousembodiments can be used with a variety of matrices including those wherethe most pronounced contamination has been encountered, including avariety of nuts, including, for example, tree nuts and peanuts, andfeeds and grains including corn, corn byproducts, soybeans, rice,sorghum, wheat and barley, oilseeds and cottonseed.

Although, many of the herein examples and descriptions refer todetecting aflatoxin, other analytes can be detected and quantified in avariety of matrices using the herein described methods and devices. Forexample, some embodiments can be used to detect aflatoxin, includingaflatoxin M1 and M2, in milk. Other possible target analytes includehormones, vitamins, drugs, metabolites and their receptors and bindingmaterials, antibodies, peptides, protein, allergens, fungicides,herbicides, pesticides and plant, animal and microbial toxins may bedetermined using the present methods and apparatuses. Other analytesthat may be determinable by the disclosed methods and apparatusesinclude antibiotics, such as beta-lactams, cephalosporins, erythromycin,sulfonamides, tetracyclines, nitrofurans, quinolones, vancomycin,gentamicin, amikacin, chloramphenicol, streptomycin and tobramycin,toxins, such as mycotoxins, vomitoxin and drugs of abuse, such asopioids and the like, as well as the metabolites thereof.

A lateral flow strip upon which various reagents and/or sample areapplied can be wholly or partially porous or bibulous so that a mobilephase can flow on or through the strip. The strip can also be wholly orpartially of a material, for example nitrocellulose, that can bindproteins. A variety of materials can be used in various portions of thestrip including natural or synthetic materials including cellulosicmaterials such as paper, cellulose and cellulose derivatives such ascellulose acetate and nitrocellulose; fiberglass; glass fiber filter,for example WHATMAN Fusion 5 membrane (Whatman is a registered trademarkof Whatman paper Limited, Kent, England); cloth, both naturallyoccurring and synthetic; porous gels such as silica gel, agarose,dextran and gelatin; porous fibrous matrices; starch based materials,such as cross-linked dextran chains; ceramic materials; films ofpolyvinyl chloride and combinations of polyvinyl chloride-silica; POREX(Porex is a registered trademark of Porex Technologies Corp., Fairburn,Ga.) and the like. Generally, the material used in the flow streamshould allow liquid to flow on or through the strip. If a variety ofmaterials are used they can be in fluid flow communication/contact orcapable of being brought into fluid flow communication/contact. Thestrip should have sufficient inherent strength or additional strengthcan be provided by a supplemental support such as a plastic backing uponwhich porous or bibulous strip components are attached.

In an embodiment an application pad is used. The application pad is influid flow communication/contact with a first end of a test strip.Contact can be either through direct contact or through an intermediatematerial allowing flow between the application pad and other portions ofthe test strip. The fluid flow communication/contact is such that thetest sample can migrate from the application pad to the other portionsof the test strip. In addition to receiving the sample, the applicationpad can also be used to drive fluid flow along the strip. Particles thatare above a certain size may clog the strip pores or may interfere withflow due to affinity to strip components thereby causing invalid orincorrect test results or otherwise reduce test function. Theapplication pad, and other strip components, can also serve as a filterto remove, from the sample, such particles.

In another embodiment, rather than pipette a pre-measured volume ontothe strip, the test strip is arranged to be dipped into a sample toabsorb a selected amount of the sample.

Mobile phase reagents can be applied to the application pad or to otherportions of the test strip, for example a POREX strip or anitrocellulose membrane, prior to sample application. Alternatively,mobile phase reagents can be premixed with the sample prior to applyingthe sample to the strip.

When mobile phase reagents are pre-applied to the strip, application ofthe sample reconstitutes the reagents for flow to portions of the teststrip such as an area where stationary phase reagents are immobilizedonto the test strip. Stationary-phase areas of the strip can includetest zones and control zones.

Mobile phase reagents can include one or multiple receptors. Receptorscan be selected for their affinity to a target analyte. The receptor maybe any agent, for example, a receptor, enzyme, membrane protein,hormone, antibody or antibody fragment, that binds with appropriatespecificity to the analyte in the test sample to form ananalyte-receptor complex. The receptor can be detected through anattached label. Colloidal gold particles are an example of a usefullabel.

The receptor can be arranged to flow in the mobile phase and be capturedby a stationary phase reagent. Locations on the test strip wherereceptors can be captured include a test zone and control zone. Eitherthe test zone or control zone can include one or multiple capture areas.The test and/or control areas can be in a variety of configurationsincluding, as commonly described, lines, dots or other configurations.

Stationary phase capture agents can be previously immobilized onto thetest strip in either or both the test zone and control zone. Captureagent immobilization to the test strip can be through proteininteraction with the solid support or various other immobilizationtechniques known in the art. For example, nitrocellulose is employed forits protein binding capacity. Capture agent immobilization can also bethrough size limitation immobilization.

The label, for example a label bound to the receptor, can be detectedwhen captured by the immobilized capture agent. To detect multipleanalytes, a test strip can have multiple test areas with differentcapture agents on each, or combined test areas with different captureagents in the same test area, to detect analytes collectively. Multipletest areas can also be employed with the same or similar capture agentsin the same or different concentrations to detect a single analyte. Whenthe same or similar capture agents are used the multiple test areas canbe employed to increase the binding potential of the test zone. Anincreased binding potential means that more labeled receptor can bindwithin the test zone.

In an embodiment of a competitive inhibition binding test, a receptorfor the analyte is labeled with a visible marker. The receptor flowswith the sample in the mobile phase to a test zone. The capture agentimmobilized at the test zone has affinity to the receptor that is thesame or similar to that of the analyte in the test sample. Such acapture agent can be, for example, a representative analyte or analoguethereof that binds to generally the same portion, or binding site, onthe receptor as does the analyte. Due to that affinity, capture agent atthe test zone can bind receptor from the mobile phase more efficientlyif the receptor is not bound by analyte from the sample. When the samplecontains analyte, an analyte-receptor complex will form that wholly orpartially prevents capture of the receptor at the test zone. As theamount of analyte in the sample increases, less receptor is captured atthe test zone until the binding at the test zone compares only to thebackground signal.

When the binding potential at the test zone is high, the range ofdetectable analyte concentrations can be wide. When the bindingpotential at the test zone is low, the overall detection range tends tobe narrower and the maximum distinguishable concentrations tend to belower. Minimizing the test zone binding potential can, however, helpincrease test sensitivity.

One method for minimizing test zone binding potential includesminimizing the amount of receptor and/or label. Another method includesminimizing the amount of test zone capture agent. These methods can alsobe employed together to titrate the amount of binding pair(receptor/capture agent) materials to generate an assay to detect theanalyte within a defined test range.

When test zone binding potential is minimized, a smaller amount ofanalyte may result in a relatively larger ratio of analyte-receptorcomplex compared to non-complexed receptor and, therefore, relativelyless capture at the test zone. Although test sensitivity may bemaximized the detection range may be narrower as well.

A method for maximizing the available range of test zone binding is tomaximize binding potential at the test zone, for example by maximizingthe amount of receptor/label and/or maximizing the amount of captureagent. A possible problem with this approach is that results in the lowrange of sensitivity may be indistinguishable. For example, a negativesample will have maximum binding at the test zone and a low level ofanalyte may inhibit some binding at the test zone. However, so muchexcess binding potential may remain that the difference in bindingcompared to a negative sample may not be readily discernible.

One embodiment is a method for maximizing binding potential, whilemaintaining test sensitivity. Such an embodiment includes using multipletest areas, within the test zone, each test area being capable ofcapturing the receptor. The multiple test areas can each contain thesame or similar concentrations of capture agent. Alternatively, the testareas can contain different concentrations of capture agent. Forexample, the first test area can have a lower concentration of captureagent as compared to the second test area. Such a configuration canaccommodate that receptor arrives at the first test area earlier and,therefore, everything else being equal, more binding will tend to occurin the first test area compared to the second. That is, with an equalamount of capture agent at each area, there would be more receptoravailable for binding to the first test area which would result in morebinding to the first test area as compared to the second test area. Ifthat is not desirable then capture agent can be titrated accordingly.

Another method for maximizing binding potential in the test zone, whilemaintaining test zone sensitivity, includes using a wider test zone.Spreading the capture agent over a larger area, similar to usingmultiple test areas, can allow greater result discrimination and lowlevel detection.

When receptors are antibodies or fragments thereof, capture agents caninclude antigens with affinity to the antibody, including analyte,analogues thereof, or any substance exhibiting affinity to the receptorthat is similar to that of the analyte. When using multiple test areasto capture the same receptor, each area can have the same capture agent.Each area can also have a different capture agent if the differentcapture agent has affinity to the same binding sites or areas as doesthe receptor.

A control zone can be used for comparison to the one or more test areasor as a signal that the test functioned properly and is complete. Thecontrol zone can include a substance as a capture agent that hasequivalent affinity to the mobile phase receptor whether or not thereceptor is bound by analyte from the sample. For example, the controlzone can include a substance that binds to a different portion ofreceptor than does the analyte. As a result, binding of analyte toreceptor to form a complex will not significantly change the bindingaffinity of the control zone capture agent to the receptor. Particularlywhen such a control zone is on a test strip in which mobile phasecontacts the control zone after contacting the test zone, the test canbe titrated to bind less receptor in a negative sample and more receptoras the concentration of analyte in the sample increases.

When the amount of receptor in the test system has been increased thebinding potential of label to the test zone binding also increases. Asecond test area can be added that binds some of the increased potentialand thereby reduces the amount of receptor available to bind in thecontrol zone. With increasing analyte in the sample the control zonewill get correspondingly darker as less label binds to the test zone andsubsequently more label becomes available for binding in the controlzone. Employing multiple test areas within the test zone to capturereceptor can be usefully employed to allow the control zone to be usedto increase the range of detection for the test system.

In another embodiment the control zone capture agent can include acapture agent that will capture a substance, conjugated to the receptoror label, which does not react with the analyte. In an example fordetection of aflatoxin, in which aflatoxin antibody is conjugated to adetectable label, a second antibody, or other substance, can beconjugated to a different label. In this way the control zone can bedesigned to bind that second antibody that does not react with aflatoxinand, therefore, provide a consistent signal that changes little whetherthe sample is positive or negative.

Some useful control zone capture agents include antibody bindingproteins such as protein A, protein G or protein AG and recombinantforms of the same. Control zone capture agents can also include anantibody, such as an anti-species antibody, alone or in combination withother antibody binding proteins. When control zone capture agents areproteins, and a solid support that binds protein, such asnitrocellulose, is used, the capture agent can be applied directly tothe support. For improved binding to the support, the control line canalso include a protein-protein conjugate with one being the captureagent and the other being a carrier protein. Useful carrier proteinsinclude, for example, bovine serum albumin (BSA), keyhole limpethemocyanin, thyroglobulin, ovalbumin, and various synthetic polypeptidessuch as poly-L-lysine, poly-L-glutamic acid, and polyethylenimine.

In a quantitative test, the changes in the test areas, and, when acontrol zone is present, the extent of the difference between thecontrol zone and test zone or test areas, can determine the test rangedetection level of analyte. To accurately and/or numerically assess thedifferences and the binding at the control zone and test zone,particularly in a quantitative assay, a reader, such as aspectrophotometer or other reflectance/absorbance reader can be used todetect and/or measure the signal provided by a chromogen such ascolloidal gold.

There are a number of possible methods for reading a result on a strip.In a strip with only one test area and one control area the control areacan be compared to the test area. In a strip containing multiple testareas possible reading methods include: finding the difference in signalintensities between the control area and each of the test areas andadding the results; finding the difference in signal intensities betweenone test area and the control area; finding the difference in signalintensities between only one test area and control area for one set ofconditions and the other test area and the control area for another setof conditions.

Using the sum of the difference in reflectance between the control areaand the two test areas can provide a greater separation between resultsfrom a range of concentrations as compared to using only the differencebetween the control area and a single test area. Using the sum of thedifferences between the control areas and each of the two test areas,can also help to reduce testing error. For example, if an aberrantresult occurred at one or the other test areas that result can bemoderated by being combined with a second set of results. This can beparticularly useful when quantitation at a particular concentration, orwithin a range of concentrations, occurs.

For quantitation, reflectance results can be converted to aconcentration value for an amount of analyte. One method for convertingreflectance to concentration can employ fitting the data to a curveusing a formula, such as:

${{concentration} = {^{(\frac{{Result} - c}{a})} - b}},$

where a, b and c are constants determined by fitting the data to acurve.

The result is the value determined by comparing reflectance in thecontrol area to reflectance at each of the test areas. For example,intensity values at each of the test areas can be separately deductedfrom intensity value at the control area and the two differences addedtogether to arrive at the result. A look-up table can also be used toconvert the mathematical result to a concentration value for theanalyte.

In another embodiment, the test can be used not only as a quantitativetest in which a reader provides a result in, for example, parts perbillion (ppb) levels but also a qualitative result. For example, thecontrol area can be compared visually with the first test area todetermine that an analyte is present above a certain threshold level,for example above 10 ppb or above 20 ppb.

Many sample matrices, such as solid or granular materials, require anextraction of analyte into a liquid matrix before application to a solidsupport such as a test strip. For example, corn can be ground to passthrough a 20-mesh sieve. The ground sample, for example 10 grams (g) or50 g, can be extracted with 70% methanol in a 2:1 ratio (2 mL per 1 g ofsample). Other extraction solvents can also be used including, forexample, acetonitrile, ethanol or other concentrations of methanol, forexample at 50%, 60%, 80% etc. Other extraction ratios can also be used,for example 5:1 extraction.

An extraction can take place using a variety of methods including:shaking the sample in a container, mixing the sample with a stirrer, ormixing the sample with a blender.

An extract can be obtained also by using a variety of methods includingfiltering to collect the extract, allowing sample to sit to form anextract layer above the ground sample, or centrifuging a portion of thesample to obtain an extract layer and sample layer.

For lateral flow assays, the sample or, if extraction is required, thesample extract, can be mixed with a dilution buffer that allows a mobilephase to flow uniformly over the test strip and/or allows reconstitutionof the dried reagents on the test strip. The extract can be diluted by anumber of methods and a variety of possible dilution ratios of theextract with the dilution buffer. The dilution buffer can consist of,for example, BSA solution, buffer or water. When the analyte is insample liquid, such as fluid milk, the sample may not require dilutionor extraction. Dilutions or extractions, however, may still be desirablesuch as to alter the test sensitivity range or to allow consistencybetween samples.

The membrane containing the beads can be pretreated with blockingsolution that dissolves when the diluted sample is added. Thenitrocellulose membrane can also be pretreated and/or blocked.

The test area and control area placement on the strip can be varied toadjust assay time. For example, a test strip utilizing 12 minuteincubation may have the test and control areas immobilized further fromthe sample application pad as compared to a test strip using only 10, 8minute or less incubation time. To shorten the incubation time, it maybe possible to move the various areas closer to the sample applicationpoint. Examples of possible locations, measured from the application endof the test strip, include respective lines for the bottom (1^(st) testarea), middle (2^(nd) test area) and top (control area) at 13millimiter(mm)/17 mm/21 mm line spacing or 17 mm/21 mm/25 mm linespacing. Although the spacing shown is equal, equal spacing may not benecessary. The sensitivity of the assay may change depending on linepositioning and, therefore, reagent titration can be adjusted toaccommodate these differences or assay requirements.

Example 1 Lateral Flow Quantitative Method for the Detection of Zero to100 ppb Aflatoxin Test Strip Overview

In this example the test strip included nitrocellulose, a POREXmembrane, a sponge and a disposal pad all in fluid flowcontact/communication. The materials were arranged as shown FIG. 1 andsecured to a plastic backing for support. The strip assembly was encasedwithin a plastic housing in which the sponge portion of the housing wassmaller than the fully expanded sponge.

The sponge was the sample application pad. The test strips were placedin a stainless steel incubator block carved to fit the test strips andheated to 4° C. When sample, including dilution buffer, was applied tothe sponge it expanded within the confines of the housing allowingliquid sample to flow to and along the POREX portion of the strip. Asthe sample flowed along the POREX strip it contacted colloidal goldparticles with aflatoxin antibody (the receptor) bound to the surface(gold-AB), creating the mobile phase. The mobile-phase, with gold-AB andsample, flowed onto the nitrocellulose membrane material from the POREX.On the nitrocellulose membrane were two test areas and one control area.The two test areas each had concentrations of immobilized capture agentthat would bind the sites of the gold-AB in the absence of previousbinding to analyte in the sample. With aflatoxin present in a sample,sites of the gold-AB bound to aflatoxin in the sample. This bindingdecreased the gold-AB binding to the test areas and, therefore,decreased the intensity as measured by the reader at each of the testareas. Inhibition of gold-AB to capture at the test areas resulted inmore gold-AB available for binding to the control area and, therefore,the intensity of the control area increased. Control area capture agentincluded antibody binding protein.

Test Strip Detail

The AB (receptor) portion of the gold-AB was a rabbit immunoglobulincreated from injecting a rabbit with a Bacillus Thuringensis(BTI)—aflatoxin immunogen. The antibody was purified starting from 5 mlof rabbit serum. To the 5 ml of serum was added 5 ml of PIERCE bindingbuffer (Pierce is a registered trademark of Pierce Biotechnology, Inc.,Rockford, Ill.) and the mixture was added to a protein A column. Afterwashing the column, the antibody was eluted from the gel with PIERCEelution buffer. The active fractions were pooled and subjected to a 50%ammonium sulfate (29.1 grams per 100 ml at 0° C.) fractionation. Theprecipitate was collected by centrifugation and the pellet was dissolvedin 2 mM diethanolamine buffer, pH 8.0, and then the solution wasdesalted on a BIO-RAD 10DG desalting column (BIO-RAD is a registeredtrademark of BIO-RAD Laboratories, Hercules, Calif.) equilibrated with 2mM diethanolamine buffer, pH 8.0. To the desalted antibody was added 1.6ml of BIO-LYTE 6/8 AMPHOLYTE (BIO-LYTE is a registered trademark ofBIO-RAD Laboratories, Hercules, Calif.) and this sample was added to aROTOFOR (ROTOFOR is a registered trademark of BIO-RAD Laboratories,Hercules, Calif.) cell for preparative isoelectric focusing. After thefirst ROTOFOR run, fractions from about pH 7.0 to pH 7.8 were collectedand subjected to a second run on the ROTOFOR system. Fractions fromabout pH 7.0 to pH 7.7 were pooled for binding to gold beads (thelabel).

To form the gold beads, 1 ml of a filtered 40 mg/ml gold chloridesolution is added to 360 ml of boiling water in a clean one liter flask.To 35 ml of water was added 3.5 ml of 1% sodium citrate solution. Thecitrate solution was added to the gold solution while boiling. Afterrefluxing for 20 to 30 minutes the bead solution was cooled and broughtto pH 8 with potassium carbonate.

Gold-AB were prepared by combining colloidal gold particles with therabbit anti-aflatoxin antibody at 4000 U of activity per 400 ml ofcolloidal gold beads at pH 8.0 while stirring. One unit of antibodyactivity is determined by binding 1000 cpm of tritiated aflatoxin B1 ina 5 minute binding assay at 35° C. using IGSORB (IGSORB is a registeredtrademark of The Enzyme Center, Lawrence, Mass.). IGSORB includesformalin-fixed Staphylococcus aureus cells containing protein A as acapture agent for the tritiated aflatoxin-antibody complex.

The gold-AB beads were mixed at room temperature for 20 minutes and thenblocked with a blocking buffer (20 mM potassium phosphate, pH 7.2,containing 5% BSA, 0.05% Bioterge, 0.025% Tween 20 and 0.0155% PROCLIN5000 (PROCLIN is a registered trademark of Rohm and Haas Company,Philadelphia, Pa.)) for at least one hour. The beads were centrifuged at9500 rpm for 50 minutes in a SLA-3000 rotor. The bead pellet wasdissolved in water and then bead storage buffer and then diluted to anabsorbance of 0.167 per 10 microliters in 3 ml of a storage buffer.Glycerol was added to the bead solution to a final concentration of16.7%. This bead solution was further diluted to 30% with bead spraysolution (0.01 M sodium phosphate, pH 7.4, containing 10% BSA and 40%sucrose). For example 25% beads would contain 1 part beads to 3 partsbead spray solution. Storage buffer includes: 0.02M potassium phosphate,pH 7.2, containing 0.2% BSA, 0.05% BIOTERGE, 0.025% TWEEN 20 and 0.155%PROCLIN 5000.

The gold-AB was sprayed (4 lines at 0.8 μl/cm) using a BIODOT sprayeronto POREX. The POREX was pretreated with 2 mM borate at pH 7.4,containing 0.1 M NaC1, 1% sucrose, 0.0025% SDS and 0.05 mg/ml reducedglutathione and dried.

The test zone with test areas and the control zone with a control areawere sprayed onto a SARTORIUS UNISART CN-140 nitrocellulose membrane(Sartorius and Unisart are registered trademarks of Sartorius AG,Gottingen, Germany). The test and control areas were sprayed onto thenitrocellulose with a BIODOT sprayer. In this example, the test areaswere located at 17 mm and 21 mm as measured from the first end of thenitrocellulose membrane. Each area had a width of 0.6 to 1.0 mm. Thetest area spray solution contained BSA conjugated to an aflatoxin B1derivative (BSA-B1). In this example, the 21 mm line contained a higherconcentration of the conjugate (0.0525 mg/ml of BSA-aflatoxin for thefirst test area and 0.084 mg/ml for the second test area).

To prepare the test area spray solution aflatoxin BSA-B1 was added to 10mM sodium phosphate buffer, pH 6.95, containing 20% sucrose and PROCLIN5000. To make the BSA-B1 conjugate, aflatoxin B1 oxime was converted toan amino derivative by using carbodiimide chemistry with 1,3diamino-2-hydroxypropane. This amino-aflatoxin derivative was thenconverted to a sulfhydryl compound with 2-iminothiolane and reacted withsulfo-SMCC activated BSA to make the conjugate. The conjugate, at thedilutions specified above, were sprayed in a line onto nitrocellulose at0.8 μl/cm using a BIODOT sprayer.

The control area was sprayed at 25 mm from the bottom of thenitrocellulose at 0.5 to 1.0 mm width. The control area spray solutionincluded protein A conjugated to BSA (BSA-PA) at a ratio of 0.6 to 2molecules of protein A to BSA. The control area spray solution included0.42 mg/ml protein.

To prepare the control area reagent 200 mg of protein A was dissolvedinto 3 ml of 0.16 M borate buffer, pH 8.1, containing 2 mM EDTA.Sulfo-SMCC was dissolved in DMSO and 3.2 mg was added to the protein Asolution. This mixture was incubated with stirring for 1.5 hours andthen cooled. The protein A-activated S-SMCC reaction was added to 2.4 mlof cold 0.4 M sodium phosphate buffer, pH 5.9, containing 2.1 grams ofBSA. The solution pH was adjusted to 6.5-7.0 and reacted overnight at 4°C. with stirring. The reaction was desalted using a high prep desaltingcolumn against 10 mM phosphate buffer, pH 6.9, containing 50 mM NaCl and4% sucrose to a final protein concentration of 50 mg/ml.

For spraying as a control area, the reaction was diluted 40 fold in 10mM sodium phosphate buffer, pH 7.2, containing 5% sucrose and PROCLIN5000. The area was sprayed in a line onto nitrocellulose, using a BIODOTsprayer, at 1.0 μl/cm and dried. The control area was sprayed and driedat the same time as the test areas.

Results were read using a ROSA reader to read reflectance on the strip.The single control area intensity was compared to each of two test areaintensities and the results were summed.

Results

In Table 1, B (bottom) and M (middle) are the first and second testareas and T (top) is the control area, respectively. The results,intensity values determined using a ROSA Reader, were as follows:

TABLE 1 Conc. Result ppb B M T T − B T − M Sum (T − B) + (T − M) 0 34963164 2102 −1394 −1062 −2456 5 3806 3812 3175 −631 −637 −1268 10 30053137 3266 261 129 390 20 2466 2823 3269 803 446 1249 100 1742 2591 40432301 1452 3753The curve of the data can be represented by the following formula forcalibration of the reader and conversion of reader results into a ppbvalue.

${{ppb} = {^{(\frac{{Result} - c}{a})} - b}},$

where a=1832; b=3; c=−4654.The constants a, b and c are determined by a curve fit for a model dataset and the result is the value from the equation [T minus B plus Tminus M] from Table 1.

Example 2

The absolute result range can be adjusted, for example so that resultsabove a certain threshold provide results in the positive number range,by adjusting the test areas and control area binding potential. Table 2below is an example of results where samples above 10 ppb provide apositive intensity value showing that the test is not limited to justdetermining a quantitative value.

TABLE 2 Concentration ppb Result 0 −2500 2.5 −1200 5 −1000 10 0 20 120037.5 2200 50 2700 75 3400 100 4000In the above table the overall spread between 0 ppb (−2500) to 100 ppb(4000) is 6500.

Example 3

In this example various methods for reading and interpreting testresults are compared, for example in the case where one test area isused to calculate an overall intensity value and where two tests areasare used to calculate an overall intensity value. results. Table 3results are, in most cases, the average of three results. The firstcolumn shows parts per billion (ppb) concentration of aflatoxin. Thesecond column shows results of a one test area/one control area testwith result interpretation by comparison of test area to control area(difference in intensity value between control area and test area). Thethird column shows results from a two test area/one control area stripwith result interpretation by utilizing only the bottom test area resultfor comparison to the control area and ignoring the middle area result.The fourth and fifth columns show results converted to differences from0 ppb result. Dilutions for one test area test were 0.035 mg/ml BSA-B1for test area and 0.25 mg/ml BSA-PA for control area. Dilutions for twotest area were 0.035 mg/ml BSA-B1, 0.07 mg/ml BSA-B1 and 0.33 BSA PA forbottom/middle/top areas, respectively. Results show an increase spreadin the two test area/one control area test (3266) when only the bottomtest area is used as compared to single test area test (2314) as aresult of increasing the test zone binding capacity with the addition ofthe second BSA-B1 test line.

TABLE 3 2 test area 1 test area line 2 test area line Conc. ppb 1 testarea bottom only diff 0 ppb diff 0 ppb 0 −973 −1104 0 0 4.3 −580 −689393 415 9.1 −129 43 844 1147 12.5 129 495 1102 1599 25 723 1045 16962149 25.7 502 1212 1475 2316 50 1005 1603 1978 2706 100 1341 2162 23143266

Table 4 compares results using one test area/one control area test withdoubling the difference between the control area and test areaintensities (second column), to results using a two test area/onecontrol area strip calculating the sum of the difference between thetest area from the control area (third column). Results show an increasespread in the two test area/one control area test. The fourth and fifthcolumns show the results converted to differences from zero. The teststrip dilutions were, as described above, 0.035 mg/ml BSA-B1, 0.07 mg/mlBSA-B1 and 0.33 BSA PA for bottom/middle/top areas, respectively, forthe two test area test and 0.03 mg/ml BSA-B1 for test area and 0.25mg/ml BSA-PA for control area for one test area test. Although thespread is increased by doubling the difference of the control area andtest area for the one test area result, error in measurement is alsoincreased.

TABLE 4 2 × diff 2 2 test area sum diff from 0 diff from 0 Conc. ppbtest area of differences 1 test area 2 test area 0 −1946 −2856 0 0 4.3−1160 −1558 786 1299 9.1 −258 −1049 1688 1807 12.5 258 −385 2204 2471 251446 724 3392 3580 25.7 1004 999 2950 3855 50 2009 1362 3955 4219 1002683 2256 4629 5112

Table 5 shows individual area intensities at each of the test andcontrol areas in a one test area/one control area test compared to thetwo test area test/one control area test. The fourth column shows thedifference in intensity between the top and bottom areas in the one testarea test and the eighth column shows the sum of the difference betweenthe intensity results between the top/middle and top/bottom areas.

TABLE 5 1 test area 2 test area Conc. ppb Top Bottom Result top middlebottom Result 0 3206 4179 −973 2111 3863 3214 −2856 4.3 3476 4056 −5802590 4237 3279 −2337 9.1 3039 3168 −129 3065 4158 3022 −1049 12.5 34143285 129 3186 4067 2691 −385 25 3361 2638 723 2942 3263 1897 724 25.73170 2668 502 3352 3565 2139 999 50 3505 2689 815 3162 3403 1560 1362100 3478 2137 1341 3354 3260 1191 2256

Example 4

The area spacing described in Example 1 (17, 21, and 25 mm—respectively,for bottom/middle/top lines) was compared with the area spacing at 13,17 and 21 mm bottom/middle/top, respectively. Both sets of areas weresprayed with test area 1 (bottom area)/test area 2 (middle area)/controlarea (top area) having respective concentrations of 0.0525 mg/ml BSA-B1,0.084 mg/ml BSA-B1 and 0.42 mg/ml BSA-PA abd tested with 25%, 4000 Ubeads.

Incubation at 40° C., in a stainless steel incubator block carved to fitthe test strips, for 10 minutes for the 13, 17 and 21 mm strip and 12minutes for the 17, 21 and 25 mm strip. At 10 minutes incubation withthe 17, 21 and 25 mm strip, the spread in results (difference betweencontrol and test area) from 0-100 ppb as compared to the 12 minuteincubation, was lower, therefore, the incubation time was increased toallow more time for the beads to flow across the test strip. When theareas were moved closer to the beads (13, 17 and 21 mm) the flow acrossthe test and control zones is quicker so that a shorter assay time ispossible.

TABLE 6 17 mm/21 mm/25 mm area spacing Conc. Ppb 10 minute incubation 12minute incubation 0 −2501 −2502 10 261 668 100 3083 3830

TABLE 7 13 mm/17 mm/21 mm area spacing Conc. Ppb 10 minute incubation 12minute incubation 0 −2462 −2150 10 961 881 100 4941 4879

Example 5

Test results were compared using the area spacing of 13, 17 and 21 mmwith 10 minutes incubation to the same area spacing with 8 minutesincubation, both at 40° C. in a stainless steel incubator block carvedto fit the test strips. Areas were sprayed with 25%, 4000 U beads andtest area 1 (bottom area)/test area 2 (middle area)/control area (toparea) respective concentrations are, respectively of 0.057 mg/mL BSA-B1,0.091 mg/mL BSA-B1, and 0.417 mg/mL BSA-PA, respectively.

Results in the following tables show that 8 minute results or 10 minuteresults are possible and that incubation at room temperature for anadditional 2 minutes had little impact on sensitivity. Although theexamples used incubation at 40° C., other temperatures can also be used.

TABLE 8 13 mm/17 mm/21 mm area spacing with a 10 minute incubation Conc.ppb Top area Middle area Bottom area Result 0 1999 4214 3933 −4150 52651 3996 3396 −2091 10 3089 3889 3062 −774 20 3494 3558 2528 901 503866 2991 1671 3069 100 3749 2479 992 4027 200 4263 2368 739 5420

TABLE 9 13 mm/17 mm/21 mm area spacing with an 8 minute incubation Conc.ppb Top area Middle area Bottom area Result 0 1740 4019 3913 −4452 52521 3959 3491 −2409 10 3094 3771 3196 −779 20 3141 3440 2417 425 503524 2731 1425 2892 100 3975 2743 1275 3933 200 4170 2131 751 5459

TABLE 10 13 mm/17 mm/21 mm area spacing with an 8 minute incubation plus2 minutes Conc. ppb Top area Middle area Bottom area Result 0 1941 42114095 −4423 5 2892 4170 3630 −2016 10 3591 4030 3441 −289 20 3421 35182465 860 50 3752 2769 1420 3315 100 4209 2823 1298 4297 200 4442 2153764 5967

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a test strip apparatus 8, comprised of nitrocellulosemembrane 3, POREX 7, sample application pad 1, and disposal pad 6attached to solid support 2. Sample is contacted to sample pad-sponge 1.Sample flows from sample application pad 1 to POREX 7 containing mobilephase receptor. A portion of receptor will bind analyte from the sampleand flow along the nitrocellulose membrane 3 to test areas 9 and 4. Aportion of receptor unbound by sample analyte will bind to the testareas. Remaining unbound receptor and analyte-receptor complex will flowto control area 5. A portion of said unbound receptor and a portion ofanalyte-receptor complex, will bind at the control area. Remainingunbound receptor or analyte-receptor complex will flow the absorbent pad6. The control area is compared to the test areas to determine a result.Although not clear from FIG. 1, the two test areas and one control areacan be evenly spaced from one another or spaced in other variations.

FIG. 2A is a graph relating intensity of color development on a teststrip to aflatoxin concentration in samples. The bottom test area of thetest strip, indicated with a diamond shape on the graph, is the firsttest area contacted by the mobile phase in the flow on the strip. Themiddle area, indicated with a square shape, is the second test areacontacted by the mobile phase in the flow on the strip. The top area,indicated with a triangle shape, is the control area and is the lastarea contacted by the sample in the flow on the strip. The result line,indicated by a circular shape, shows the results which are calculated bymeasuring the difference in signal intensity between each test area andthe control area and summing the differences. The bottom area, at 0 ppb,has the highest intensity and the middle area has the second highestintensity. As the concentration of analyte, in this example aflatoxin,increases there is a steady loss in intensity at both the bottom areaand middle area (the test areas), reflecting the inhibition of labeledreceptor from binding to the test areas. Results to 50 ppb showrelatively constant inhibition at the test areas. That is, in this dataset, there is little difference in test area intensities between 50 ppband 100 ppb while the control area intensity changes significantly from50 ppb to 100 ppb, providing the test result difference from 50 ppb to100 ppb. As is shown by the results line (calculated by deducting thebottom area intensity from the top area intensity and deducting the toparea intensity from the middle area intensity, and then adding the twodifferences), the intensity difference between each of the test areasand the control area provides the sensitivity to distinguish, in thisparticular test strip, between aflatoxin concentrations between 0 ppband 100 ppb.

FIG. 2B is a graph of the results from the same experiments as in FIG.2A with additional concentrations included. Additional data points showthat the results are corrected for variations in area intensity that mayhave resulted from flow differences within the test strip or from samplevariation resulting from different sources of raw materials tested. Forexample, the results (circle) at around Intensity/Result −1000 arecorrected from the aberrant results in the control and test areas.

FIG. 3A is a graph of the results from experiments with a single testarea and control area using a 0.035 mg/ml conjugate-BSA-B1 on the testarea and 0.025 mg/ml BSA-PA) on the control area. The result area(top-bottom), indicated by the triangle, reflects lower sensitivity thanthe result line in FIGS. 2A and 2B. That is, there is significantly lessspread (difference between control area and test area), in the one testarea test, at varying concentrations as compared to the two test areatest.

FIG. 3B demonstrates how changing the concentration of the top area,from 0.025 mg/ml to 0.016 mg/ml BSA-PA, indicated by the diamond,impacts test results, indicated by the triangle. The top area intensityat 0 ppb starts at 2000, rather than 3000 (as in FIG. 3A). As with FIG.3A, the result line, indicated by the triangle, is less sensitive thanthe result line in FIG. 2A and FIG. 2B. There is, therefore,significantly less spread (difference) between results at varyingconcentrations as compared to using two test areas. The comparisonbetween FIG. 3A and FIG. 3B shows that changing the concentration at thecontrol area shifts the lines—the top area intensity at 0 ppb (yintercept) starts at 2000, rather than 3000. Changing the concentrationat the control area, however, does not significantly change the spreadbetween results at various concentrations. For example, in FIG. 3A, theresult at 0 ppb is −1000 and the result at 100 ppb is 2000, providing a3000 point spread. In FIG. 3B the result at 0 ppb is −2000, and theresult at 100 ppb is 1000, again providing a 3000 point spread. This iscompared to, in FIG. 2A and FIG. 2B, an approximately 6000 point spreadbetween 0 ppb and 100 ppb. Thus, the second test area can provide asignificant increase in spread and, therefore, can provide more accuracyfrom concentration to concentration. That is, a larger spread allows agreater margin of error without affecting test results. The largermargin of error is particularly important, for example, when samples andresults are variable and when quantitation is required.

FIG. 4 is a graph comparing results from a single test area/control areatest for aflatoxin with a two test area/single control area test foraflatoxin, where only the first test area was used to calculate results.This graph demonstrates that even when the second test area is not usedto calculate results, the added binding capacity created by the presenceof the second test area can help increase the detection range of thetest. The inconsistency in the data—the one test area test shows a dropin intensity from 25 ppb to 25.7 ppb—demonstrates the importance of alarger spread when testing multiple sample lots.

1. A method for the analysis of a liquid sample for the presence of ananalyte comprising the steps of: a) contacting the sample with areceptor to form a mobile phase, the receptor characterized by anability to bind to the analyte to provide, in the mobile phase, areceptor-analyte complex; b) contacting the mobile phase with a solidsupport, the solid support configured to allow lateral flow of themobile phase to a first test area, a second test area and a controlzone, the first test area, the second test area and the control zoneconfigured on the solid support in a spaced apart relationship and eachcomprising a capture agent immobilized on the solid support, the firstand the second test area capture agents characterized in that thecapture agent has greater binding affinity to the receptor than to thereceptor-analyte complex, the control zone capture agent characterizedin that the affinity to the receptor is equivalent to the affinity tothe analyte-receptor complex, the solid support configured to allow themobile phase to flow from the first test area to the second test area onthe solid support and from the second test area to the control zone onthe solid support; c) prior to, currently with, or subsequent to steps(a) and (b), tagging the receptor with a label, the label characterizedin that it provides a detectable signal when the receptor is bound toany of the first test area capture agent, the second test area captureagent and the control zone capture agent; and d) measuring the intensityof the detectable signal at each of the first test area, the second testarea and the control zone, wherein the intensity of the detectablesignals in the test areas are inversely related to a concentration ofthe analyte in the sample.
 2. The method of claim 1 wherein the signalintensities are measured by an electronic test instrument and whereinthe instrument: (i) measures intensity at each of the two test areas andthe control zone; (ii) sums the intensity at the two test areas; and(iii) compares the sum of the signal intensities at the first test areaand the second test area with the signal intensity at the control zoneto determine a test result.
 3. The method of claim 2 further comprisingusing the test result to determine the concentration of the analyte inthe sample.
 4. The method of claim 3 wherein the concentration of theanalyte in the sample is determined by a comparison with a predefinedtest parameter.
 5. The method of claim 1 wherein the signal comprises avisible signal and wherein the label comprises a gold particle.
 6. Themethod of claim 1 wherein a test result is determined by comparing thesignal intensities at the first test area and the second test area withthe signal intensity at the control zone.
 7. The method of claim 1wherein a test result is determined by comparing the signal intensitiesat one of either the first test area or the second test area with thesignal intensity at the control zone.
 8. The method of claim 1 wherein atest result, related to the signal intensity, is provided by anelectronic instrument, and wherein the instrument: (i) measures thesignal intensities; (ii) calculates the difference between the firsttest area intensity and the control zone intensity; (iii) calculates thedifference between the second test area intensity and the control zoneintensity; (iv) stuns the results of (ii) and (iii) to arrive at thetest result; (v) determines the concentration of analyte in the sampleby comparing the test result to a predetermined value; and (vi) displaysthe analyte concentration.
 9. The method of claim 8 wherein the testresult is a digital result.
 10. The method of claim 8 wherein theinstrument comprises a reflectance reader.
 11. The method of claim 1wherein the receptor is labeled prior to contact with the liquid sample.12. The method of claim 1 wherein the first test area capture agent isthe same as the second test area capture agent.
 13. The method of claim12 wherein the first test area capture agent and the second test areacapture agent are in the same concentration.
 14. The method of claim 12wherein the first test area capture agent and the second test areacapture agent are in different concentrations.
 15. The method of claim 1wherein the first test area capture agent and the second test areacapture agent are representative analyte or analog thereof.
 16. Themethod of claim 1 wherein the first test area capture agent and thesecond test area capture agent are different capture agents.
 17. Themethod of claim 1 wherein the analyte comprises a mycotoxin.
 18. Themethod of claim 1 wherein the liquid sample comprises a liquid extract.19. The method of claim 8 wherein the concentration is determined usinga formula, the formula comprising:${concentration} = {^{(\frac{{Result} - c}{a})} - b}$ where a, b, and care constants.
 20. The method of claim 3 wherein the concentrationcomprises an approximate concentration.
 21. The method of claim 8wherein the concentration comprises an approximate concentration. 22.The method of claim 19 wherein the concentration comprises anapproximate concentration.